Compositions and methods for screening antimicrobials

ABSTRACT

A method for screening compounds for antimicrobial activity is described that utilizes bacterial protein—protein binding in vitro. The method may be performed using immobilized elements and the immobilization may be carried out using a variety of immobilization means (e.g., columns, beads, adsorbents, nitrocellulose paper, etc.) in order to screen large libraries of compounds.

This is a continuation of application(s) 09/184,826 filed on Nov. 2,1998 now U.S. Pat. No. 6,248,543 which is a divisional application of08/651,818 filed on May 21, 1996 issued AUG. 21, 1996 U.S. Pat. No.5,948,889.

FIELD OF THE INVENTION

The invention relates to screening compounds for antimicrobial activity,and, more particularly, to using bacterial proteins in vitro to detectcompounds that interfere with cell division.

BACKGROUND

Antimicrobials are developed on the principle of selective toxicity.That is to say, antimicrobials, while toxic to the microorganism, mustnot be toxic to the patient. The selective toxicity of these drugs isusually relative, rather than an absolute. This means simply that mostdrugs are given to patients in concentrations that are tolerated by thepatient, but are lethal or damaging to the microorganism; higher doseswould be toxic to the patient and are avoided.

Selective toxicity is often a reflection of the presence of specificreceptors present on the microorganism, but lacking in the host system.Other means to achieve selective toxicity commonly rely on theinhibition of biochemical events essential to the microorganism but notthe host. As the physiology, structure, and biochemical systems ofinfectious agents and their hosts are usually quite different,antimicrobial development often relies on these differences.

Although the mechanisms of action of many antimicrobials are not wellunderstood, the five major categories of action include inhibition ofcell wall synthesis, inhibition of cell membrane function, inhibition ofprotein synthesis, inhibition of nucleic acid synthesis, andinterference with intermediary metabolism. (See e.g., W. K. Joklik etal., [eds.], Zinsser Microbiology, 18th ed., Appleton-Century-Crofts,Norwalk, Conn., [1984], p. 193). For example, penicillin, like allβ-lactam drugs, is a compound which selectively inhibits bacterial cellwall synthesis. The initial step in the mechanism of action of theseβ-lactam drugs involves the binding of the drug to cell receptors knownas “penicillin-binding proteins” (“PBP”). There are from 3-6 PBPs, withmolecular weights ranging from 4-12×10⁵; some of these PBPs aretranspeptidation enzymes. (Jawetz, Melnick & Adelberg's MedicalMicrobiology, 19th ed, Appleton & Lange, Norwalk, Conn. [1991], p. 150).After binding to the PBP, the drug inhibits the transpeptidationreaction and synthesis of peptidoglycan in the organism's cell wallmaterial is blocked. This results in the eventual triggering of anautolytic cascade which leads to cell lysis.

Because of their relatively high concentration of peptidoglycan,gram-positive organisms tend to be much more susceptible to the effectsof penicillin and other β-lactams than gram-negative organisms.Importantly, because they affect cell wall synthesis, penicillin and theother β-lactams are only effective against actively growing and dividingcultures. However, one of the benefits of these β-lactam drugs is thatanimal cells do not have peptidoglycan; consequently, such drugs areremarkably non-toxic to humans and other animals.

Some organisms are naturally resistant to penicillin and the otherβ-lactams due to their lack of PBPs, the inaccessibility of the PBPs dueto the presence of permeability barriers, the failure of autolyticcascades to be activated following binding of the drug, or the lack ofpeptidoglycan in the cell wall (e.g., the mycoplasmas, L-forms, andmetabolically inactive bacteria). Unfortunately, following years of useto treat various infections and diseases, penicillin resistance hasbecome increasingly widespread in the microbial populations that werepreviously susceptible to the action of these drugs. Some microorganismsproduce β-lactamase, an enzyme which destroys the antimicrobial itself,while some microorganisms have undergone genetic changes which result inalterations to the PBPs, such that the drugs will no longer effectivelybind to the receptors; still other organisms have evolved in a mannerthat prevents the lysis of cells to which the drug has bound. In thislatter scenario, the drug has inhibited the growth of the cell, but itis not killed. In some circumstances this appears to contribute to therelapse of disease following premature discontinuation of treatment, assome of the cells remain viable and may begin growing once theantimicrobial is removed from their environment.

The development of tolerance and resistance to antimicrobials representsa significant threat to the ability to treat disease. Many factors havecontributed to this increased observance of resistant strains, includingover-use and/or inappropriate administration of antimicrobials, thecapability of many organisms to exchange genetic material which confersresistance (i.e., R plasmids), and the relatively rapid mutation rateobserved with many bacteria, which allows for selection of resistantorganisms.

One well-documented example which highlights the problems withdevelopment of penicillin resistance involves Streptococcus pneumoniae,a gram-positive organism. Initially, the introduction of penicillin totreat S. pneumoniae resulted in a significant decrease in the mortalitydue to this organism. However, S. pneumoniae remains of great concern,as it is one of the agents most frequently associated with invasiveinfections; it is the most common cause of bacterial pneumonia andotitis media; it is the second most common cause of bacterialmeningitis; and it is the third most common isolate from blood cultures.(J. F. Sessegolo et al., “Distribution of Serotypes and AntimicrobialResistance of Streptococcus pneumoniae Strains Isolated in Brazil From1988 to 1992,” J. Clin. Microbiol., 32:906-911 [1994]). Thus, thedevelopment of antimicrobial resistance in this organism is of greatcause for concern.

The first report of pneumococci with decreased susceptibilities topenicillins occurred in 1967. Since this initial report out ofAustralia, additional strains with decreased susceptibilities have beenreported worldwide. Additionally, resistance to penicillin alternatives,such as chloramphenicol, erythromycin, tetracycline clindamycin,rifampin, and sulfamethoxazole-trimethoprim has been reported, often inconjunction with penicillin resistance. Multiple-antimicrobialresistance in pneumococci was first reported in 1977. Since this initialreport out of South Africa, multi-drug resistant strains have beenreported in several countries, including Spain, Italy, France, Belgium,Hungary, Pakistan, Czechoslovakia, Canada, the United Kingdom, and theUnited States. (Sessegolo et al. supra, at 906).

In a survey conducted in Brazil, of 42 serotypes among 288 S. pneumoniaestrains isolated during 1988-1992, Sessegolo et al. reported thatdecreased susceptibility to penicillin was detected in 26.7% of thestrains. In addition, 35.9% of the strains were resistant totetracycline, 29.2% were resistant to sulfamethoxazole-trimethoprim,1.5% were resistant to rifampin, 0.80% were resistant to penicillin, and0.50% were resistant to chloramphenicol. The penicillin-resistantstrains were also found to be resistant to, or exhibited decreasedsusceptibility to cephalosporins. The resistance characteristics ofthese strains were also semi-quantitated, with intermediate resistancesreported at 17.9% for penicillin, 8.7% for tetracycline, 6.7% forchloramphenicol, 6.1% for erythromycin, and 3.1% for rifampin.

Results obtained from patients in Rio de Janiero in 1981 and 1982,indicated that there was no penicillin resistance (relative or complete)in the pneumococcal isolates. However, during the period between 1988 to1992, 19.4% of the strains from the same geographic population wererelatively resistant, and 1.5% were completely resistant to penicillin.These results highlight the rapid spread of antimicrobial resistance.

Once an organism has developed resistance to a particular drug, itbecomes important that an effective replacement drug be identified. Ifthe organism develops resistance to this second drug, anotherreplacement is needed. One example of the historical development ofmultiple drug resistance is gonorrhea. Prior to the 1930′s, treatmentfor this disease usually involved mechanical means, such as irrigationand use of urethral sounds in males. In the late 1930′s, sulfonamideswere introduced and found to be effective in treating gonorrhea. After afew years, sulfonamide-resistant strains of N. gonorrhoeae wereisolated. Fortunately, by this time, penicillin was available and foundto be effective. However, by the 1970′s, many isolates of N. gonorrhoeaewere found to be penicillin-resistant. This required the use ofalternative drugs such as spectinomycin. It can be expected that thistrend will continue, with the development of strains that are resistantto sulfonamides, penicillin, spectinomycin, and other antimicrobials.

Thus, there remains a need to develop new antimicrobials. Ideally, theantimicrobial should target the physiology of the microorganism anddemonstrate selective toxicity. However, the targeting should,nonetheless, allow for antimicrobial action against a broad spectrum oforganisms. Most importantly, the antimicrobial should serve as aneffective replacement drug for multiple-drug resistant organisms.

SUMMARY OF THE INVENTION

The invention relates to screening compounds for antimicrobial activity,and, more particularly, to using bacterial proteins in vitro to detectcompounds that interfere with cell division. The present inventioncontemplates the use of the zipA gene and gene product for screeningcompounds for potential antimicrobial activity. Unlike current screeningapproaches, the screening approach of the present invention does notrequire the use of bacterial cells.

The present invention contemplates the over-expression of recombinantZipA protein that is functional, and yet free of contaminating proteintypically associated with traditional biochemical isolation techniques.The expression of recombinant ZipA protein of the present inventionrelies on the construction of vectors (e.g., plasmids) containing thezipA gene and suitable hosts for protein expression. It is not intendedthat the present invention be limited by the expression system chosenfor the expression of recombinant zipA. The present inventioncontemplates all forms and sources of expression systems (i.e., anexpression vector/host cell combination).

In one embodiment, the present invention contemplates a method forscreening compounds, comprising: a) providing: i) a test compound; ii) afirst protein, said first protein encoded by an oligonucleotidecomprising at least a portion of the zipA gene; iii) a second proteincapable of binding to said first protein; and iv) means for detectingsaid binding; b) mixing said first and second proteins in the presenceof said test compound; and c) detecting binding using said means fordetecting binding.

The method may be performed using immobilized elements and theimmobilization may be carried out using a variety of immobilizationmeans (e.g., columns, beads, adsorbents, nitrocellulose paper, etc.). Inorder to screen large libraries of test compounds (e.g., drugs, newantimicrobials, etc.), the screening assays of the present invention arepreferably conducted in a microplate format.

The present invention contemplates a variety of assay formats. In oneembodiment, said first protein (encoded by an oligonucleotide comprisingat least a portion of the zipA gene) is immobilized. In anotherembodiment, said second protein (capable of binding to said firstprotein) is imrnobilized. In one embodiment, said second protein isFtsZ. In one embodiment, said second protein is labelled (e.g.,radiolabelled).

It is not intended that the invention be limited by the means or methodof detection. For example, the detection means might be a plate reader,a scintillation counter, a mass spectrometer or fluorometer.

It is not intended that the invention be limited by the nature of testcompounds. Such compounds may be synthetic compounds or naturallyavailable compounds.

The method of the present invention is particularly useful foridentifying antimicrobials effective against bacteria. However, suchidentified drugs may have activity against other single cell andmulticellular organisms, including, but not limited to fungi, mycoplasmaand protozoa.

DESCRIPTION OF THE FIGURES

FIG. 1 schematically shows proteins involved in bacterial cell divisionthat are contemplated to be useful in the present invention forscreening antimicrobials.

FIG. 2 is a audioradiograph of a binding assay using proteinsimmobilized on nitrocellulose.

FIG. 3 schematically shows the location of the zipA gene on the E. colichromosome, a hydrophilicity plot across the ZipA protein, and aschematic representation of ZipA within the inner membrane of a cell.

FIG. 4 shows the nucleotide sequence of a portion of the E. colichromosome which contains the entire zipA gene. The nucleotide sequenceof the zipA gene is provided (SEQ ID NO:1) as well as the amino acidsequence of the ZipA protein (SEQ ID NO:2).

FIG. 5 shows the amino acid sequence of the ZipA protein homologue in H.influenzae (SEQ ID NO:3:) aligned with the amino acid sequence of theZipA protein in E. coli (SEQ ID NO:2).

FIG. 6 schematically shows the expression cloning utilized in thepresent invention.

FIG. 7 schematically shows a number of plasmid constructs containingzipa sequences.

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.” Commonlyemployed vectors include, but are not limited to, plasmids andbacteriophage vectors.

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in procaryotes usually include a promoter, anoperator (optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, the sequence “A-G-T,” iscomplementary to the sequence “T-C-A.” Complementarity may be “partial,”in which only some of the nucleic acids' bases are matched according tothe base pairing rules. Or, there may be “complete” or “total”complementarity between the nucleic acids. The degree of complementaritybetween nucleic acid strands has significant effects on the efficiencyand strength of hybridization between nucleic acid strands. This is ofparticular importance in amplification reactions, as well as detectionmethods which depend upon binding between nucleic acids.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5±0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl. (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization (1985). Other referencesinclude more sophisticated computations which take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. With “high stringency” conditions, nucleicacid base pairing will occur only between nucleic acid fragments thathave a high frequency of complementary base sequences. Thus, conditionsof “weak” or “low” stringency are often required with nucleic acids thatare derived from organisms that are genetically diverse, as thefrequency of complementary sequences is usually less.

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids which may be amplified by any amplification method,including but not limited to the polymerase chain reaction (PCR). It iscontemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample. In contrast, “background template” is used inreference to nucleic acid other than sample template which may or maynot be present in a sample. Background template is most ofteninadvertent. It may be the result of carryover, or it may be due to thepresence of nucleic acid contaminants sought to be purified away fromthe sample. For example, nucleic acids from organisms other than thoseto be detected may be present as background in a test sample.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (ie., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, which is capable ofhybridizing to another oligonucleotide of interest. Probes are useful inthe detection, identification and isolation of particular genesequences.

It is contemplated that proteins (or probes) used in the presentinvention will be labelled with a “reporter molecule,” so that isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is not intendedthat the present invention be limited to any particular detection systemor label, whether a radioisotope, enzyme and flurogenic substance orother type of molecule (e.g., biotin, etc.). There are a number ofcommercially available kits to take advantage of particular labellingschemes (one such kit being available from Amersham and described inU.S. Pat. No. 4,568,649, hereby incorporated by reference).

As used herein, the term “target” refers to the region of nucleic acidbounded by the primers used for detection and/or amplification (e.g., bythe polymerase chain reaction). Thus, the “target” is sought to besorted out from other nucleic acid sequences. A “segment” is defined asa region of nucleic acid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of K. B. Mullis U.S. Pat. Nos. 4,683,195 and 4,683,202,hereby incorporated by reference, which describe a method for increasingthe concentration of a segment of a target sequence in a mixture ofgenomic DNA without cloning or purification.

As used herein, the terms “PCR product” and “amplification product”refer to the resultant mixture of compounds after two or more cycles ofthe PCR steps of denaturation, annealing and extension are complete.These terms encompass the case where there has been amplification of oneor more segments of one or more target sequences.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleoside triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

As used herein, the term “recombinant DNA molecule” as used hereinrefers to a DNA molecule which is comprised of segments of DNA joinedtogether by means of molecular biological techniques.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotides referred to as the “5′end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. In either alinear or circular DNA molecule, discrete elements are referred to asbeing “upstream” or 5′ of the “downstream” or 3′ elements. Thisterminology reflects the fact that transcription proceeds in a 5′ to 3′fashion along the DNA strand. The promoter and enhancer elements whichdirect transcription of a linked gene are generally located 5′ orupstream of the coding region. However, enhancer elements can exerttheir effect even when located 3′ of the promoter element and the codingregion. Transcription termination and polyadenylation signals arelocated 3′ or downstream of the coding region.

As used herein, the term “an oligonucleotide having a nucleotidesequence encoding a gene” means a DNA sequence comprising the codingregion of a gene or in other words the DNA sequence which encodes a geneproduct. The coding region may be present in either a cDNA or genomicDNA form. Suitable control elements such as promoters and/or enhancers,splice junctions, polyadenylation signals, etc., may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript in eukaryotic host cells. When prokaryotes are used as thehost cell, suitable control elements may include but are not limited topromoters, operators, ribosome binding sites, and transcriptiontermination elements. When expression is desired in a prokaryotic host,the coding region employed will typically lack introns. Alternatively,the coding region utilized in the expression vectors of the presentinvention may contain endogenous enhancers/promoters, splice junctions,intervening sequences, polyadenylation signals, etc., or a combinationof both endogenous and exogenous control elements.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

The term “drug” as used herein, refers to any medicinal substance usedin humans or other animals. Encompassed within this definition arecompound analogs, naturally occurring, synthetic and recombinantpharmaceuticals, hormones, antimicrobials, antibiotics, etc.

DESCRIPTION OF THE INVENTION

The invention relates to screening compounds for antimicrobial activity,and, more particularly, to using bacterial proteins in vitro to detectcompounds that interfere with cell division. The present disclosuredescribes the isolation of the gene encoding the ZipA protein. Anexpression vector comprising a plasmid has been constructed to allow forthe protein to be functionally over-expressed in bacterial cells.However, the plasmid also is constructed to be functionallyover-expressed in other hosts. Indeed, it is not intended that thepresent invention be limited by the expression system; the presentinvention contemplates all forms and sources of expression systems.

In one embodiment, the present invention contemplates using the ZipAprotein in a cell-free assay to screen compounds for their antimicrobialactivity. The cell-free assay has inherent advantages over traditionalantimicrobial screening assays.

In another embodiment, the present invention contemplates using the ZipAprotein in a cellular assay. One cellular assay contemplated is theyeast two-hybrid system.

I. Traditional Antimicrobial Screening

To understand the limitations of cell-based screening assays (and theadvantages of the cell-free screening assay of the present invention),it is useful to consider the principles inherent for the detection ofcell growth. For example, a bacterial culture may be considered“sterilized” if an aliquot of the culture does not grow when transferredto fresh culture media (e.g., solid or liquid culture media) and placedunder suitable conditions such that growth of the organism may occur.The time period and the growth conditions (e.g., temperature) may bereferred to as an “amplification factor.” This amplification factor,along with the limitations of the detection method (e.g., visualinspection of the culture plate for the appearance of bacterialcolonies) define the sensitivity of the inactivation method. A minimalnumber of viable bacteria must be applied to the plate for a signal tobe detectable. With the optimum detection method, this minimal number is1 bacterial cell. With a suboptimal detection method, the minimal numberof bacterial cells applied so that a signal is observed may be muchgreater than 1. The detection method determines a “threshold” belowwhich the method appears to be completely effective (and above which themethod is, in fact, only partially effective).

This interplay between the amplification factor of an assay and thethreshold that the detection method defines, can be illustrated. Forexample, bacterial cells can be applied to a plate of culture media; thedetection method is arbitarily chosen to be visual inspection. Assumethe growth conditions and time are such that an overall amplification of10⁴ has occurred. The detectable signal will be proportional to thenumber of bacterial cells actually present after amplification. Forcalculation purposes, the detection threshold is taken to be 10⁶ cells;if fewer than 10⁶ cells are present after amplification, no cellcolonies are visually detectable and the inactivation method will appeareffective. Given the amplification factor of 10⁴ and a detectionthreshold of 10⁶, the sensitivity limit would be 100 bacterial cells; ifless than 100 viable bacterial cells were present in the originalaliquot of the bacterial culture after the sterilization method isperformed, the culture would still appear to be sterilized.

Such a situation is common for bacterial growth assays. The sensitivityof the assay is such that viable bacterial cells are present but theassay is unable to detect them.

Antimicrobial susceptibility testing using cell growth assays is onemethod to determine the effectiveness of an antimicrobial against amicroorganism. Antimicrobial susceptibility is measured in vitro, inorder to determine the toxicity (i.e., potency) of the antimicrobial insolution, estimate its concentration in body tissues or fluids, anddetermine the sensitivity of a given organism to known concentrations ofthe antimicrobial. There are various factors which may significantlyaffect the activity of antimicrobials in vitro. Such factors as the pHof the testing environment, the medium upon which the microorganismswere grown, the stability of the antimicrobial, the inoculum size,length of exposure to the antimicrobial, and the metabolic activity ofthe microorganisms must be taken into account during the in vitrotesting of antimicrobial susceptibility. (See e.g., G. F. Brooks, etal., Jawetz, Melnick & Adelberg's Medical Microbiology, 19th ed,Appleton & Lange, Norwalk, Conn. [1991], p.155).

In many cases, it is possible to quantitate the amount or concentrationof antimicrobial required to kill or inhibit microorganisms, withoutharming the patient. Quantitation becomes critical in some situations,as illustrated by the situation where an organism that previously wassusceptible to low concentrations of an antimicrobial developsresistance to that antimicrobial. In addition, it is often not possibleto predict the susceptibility of a particular organism or isolate to aparticular antimicrobial. (See e.g., J. J. Jorgensen and D. F. Sahm,“Antimicrobial Susceptibility Testing: General Considerations,” in P. R.Murray et al. [eds.], Manual of Clinical Microbiology, 6th ed., ASMPress, Washington, D.C., [1995], p. 1277). For all these reasons, thepresent invention is not limited to a cell-based screening method.Rather, the present invention allows for a cell-free detection systemthat does not have the drawbacks associated with measuring parameters oflive cells.

II. Proteins Useful in the Present Invention

In one embodiment, the present invention contemplates a cell-free methodfor screening compounds for antimicrobial activity involves usingbacterial proteins in vitro to detect compounds that interfere with celldivision. Thus, the present invention contemplates bacterial proteinsinvolved in cell division (see FIG. 1) as proteins useful in compoundscreening. One such protein is the essential division protein FtsZ,which is very well conserved among different species.

Immediately before the start of cell division, FtsZ becomes concentratedat the inner membrane into a ring-like structure at the prospectivedivision site. (See FIG. 1). During septation, the diameter of the FtsZring becomes smaller as it remains at the leading edge of theinvaginating cell wall. Although the FtsZ-ring is thought to be crucialfor cell wall invagination, its precise role is not known. In oneattractive hypothesis, the ring is a contractile element which causesthe cytoplasmic membrane to move inwards. This, in turn, could triggerthe septum specific murein synthetase PBP3 (Fts1) to back up the inwardmoving membrane with a rigid murein layer.

Experiments performed in vitro with purified FtsZ have shown that theprotein is a GTPase with the ability to form large polymers in anucleotide-dependent fashion. This is consistent with the idea that theFtsZ ring contains one large, or multiple smaller, polymer (s)consisting of GTP/GDP-bound FtsZ subunits. The FtsZ peptides from thethree bacterial species of known sequence share a 13 amino-acid segmentthat is completely conserved and that includes a 7 amino-acid stretchalmost identical to the highly conserved sequence among the α-, β- andγ-tubulins of eukaryotic cells. A single amino-acid substitution withinthe tubulin signature sequence of FtsZ leads to a failure to initiateseptum formation in cells grown at elevated temperatures.

While it is not intended that the present invention be limited byprecise mechanisms, it is believed that FtsZ needs to interact withseveral different molecules that play specific roles in one or more ofthe cell division processes. Genetic studies have suggested possibleinteractions between FtsZ and several other proteins. Moreover, there issolid physical evidence for an interaction between FtsZ and FtsA.Indeed, FtsA can be co-purified with FtsZ and vice versa.

To address the possibility that other proteins interact with FtsZ, thepresent inventors searched for proteins with affinity to FtsZ. In oneapproach, a FtsZ derivative (HFKT-FtsZ) was made which carries at theN-terminus a histidine-tag (the his-tag is useful for purification bymetal chelate affinity chromatography such as that described in U.S.Pat. No. 5,310,663, hereby incorporated by reference) as well as asubstrate site for heart muscle kinase. The purified protein wasradiolabeled in vitro by incubation with kinase and [γ³²P]ATP and usedas a probe on Western blots. In this assay, HKFT-FtsZ bound specificallyto one minor protein band present among the E. coli proteins found inthe insoluble (P200) fraction (and not to any bands in the soluble(S200) fraction) corresponding to a species with an apparent MW of 50 Kd(see FIG. 2, middle panel), which the inventors have named “ZipA.”Binding of the protein was specific for the FtsZ portion of theHKFT-FtsZ probe, since HKFT-tagged derivatives of several other proteinsfailed to bind to the 50 Kd band (the left-hand panel of FIG. 2 showsthat radiolabelled HKFT-MinE binds Omp but not ZipA, thereby serving asa negative control) and since native FtsZ competes for binding [theright-hand panel of FIG. 2 shows decreased signal when native,unlabelled FstZ (indicated as “+FtsZ”) is included in the assay].

ZipA was exclusively present in the insoluble fraction of broken cells.Treatment of this fraction with urea did not release ZipA, whereastreatment with either Sarkosyl or Triton X-100 efficiently solubilizedthe protein. This suggests that ZipA is an integral inner-membraneprotein. While an understanding of the precise role of ZipA in celldivision is unnecessary for the practice of the present invention, FIG.1 schematically sets forth a possible arrangement for ZipA in relationto other known cell division proteins.

The gene for ZipA (zipA) was isolated by expression cloning from a λgt11library, and shown to be a previously unidentified gene at 52 minutes onthe E. coli chromosome. (See FIG. 3). Overexpression of the zipA openreading frame (ORF) in a T7 RNA polymerase based system led tooverproduction of a single protein which migrated as a 50 Kd species,and which readily bound FtsZ in the assay described above. The predictedprimary structure of ZipA shows aa hydrophobic N-terminus, and anabundance of proline residues (12.5%). The calculated MW is 36.4 Kdrather than 50 Kd indicating that the protein migrates aberrantly inSDS-PAGE gels. Database searches did not reveal any known proteins withsignificant similarity to ZipA. DNA fragments carrying the complete zipAgene could not be cloned on high copy number vectors, but could bemaintained when carried on phage M13 or a low copy number plasmid. Thenucleotide sequence of the zipA gene (SEQ ID NO:1) is shown in FIG. 4.

The ZipA protein should form an attractive basis for antimicrobialcompound screens for several reasons, including but not limited to: 1)ZipA is essential, cells that lack sufficient ZipA activity die; 2) ZipAbinds to FtsZ, even when either of the two proteins is (partially)denatured (in fact, ZipA was discovered by an affinity blottingtechnique in which radiolabeled FtsZ was used to probe Western blots ofwhole cell extracts that had been treated by boiling in detergent); 3)soluble fragments and derivatives of ZipA retain the ability to bindFtsZ [ie., a portion or fragment of the ZipA protein can be expressed(defined as the 39-328 peptide, see FIG. 4) as soluble protein (usingthe sequence of the zipA gene generated by digestion at the second PvuIsite within the coding region) and this portion binds FtsZ]; 4)HFTK-ZipA will bind to native FtsZ as well as immobilized FtsZ; and 5)labelled derivatives of ZipA (e.g., in which a portion of the protein isfused to Green Fluorescent Protein) retain the ability to bind FtsZ.

The above-described features should make clear the great flexibility inthe design of large screens for compounds that either interfere with theability of FtsZ and ZipA to interact, or that bind to ZipA per se.

Antibodies

The present invention contemplates the use of antibodies, including theuse of antibodies, in the screening assays. Such antibodies can be usedeither as a positive control (i.e., as antibodies that block interactionof ZipA with another protein) or as a binding partner for otherproteins.

The antibodies directed to such proteins as ZipA and FtsZ or theZipA:FtsZ binding complex (“primary antibodies”) may be monoclonal orpolyclonal. It is within the scope of this invention to include anysecondary antibodies (monoclonal or polyclonal) directed to the primaryantibodies discussed above. Both the primary and secondary antibodiesmay be used in the detection assays or a primary antibody may be usedwith a commercially available anti-immunoglobulin antibody. An antibodyas contemplated herein includes any antibody specific to any region of acell division protein.

Both polyclonal and monoclonal antibodies are obtainable by immunizationwith the protein and either type is utilizable for immunoassays. Themethods of obtaining both types of antibodies are known in the art.Polyclonal antibodies are less preferred but are relatively easilyprepared by injection of a suitable laboratory animal with an effectiveamount of the purified protein, or antigenic parts thereof, collectingserum from the animal, and isolating specific antibodies by any of theknown immunoadsorbent techniques. Although antibodies produced by thismethod are utilizable in virtually any type of immunoassay, they aregenerally less favored because of the potential heterogeneity of theproduct.

The use of monoclonal antibodies in an immunoassay is particularlypreferred because of the ability to produce them in large quantities andthe homogeneity of the product. The preparation of hybridoma cell linesfor monoclonal antibody production derived by fusing an immortal cellline and lymphocytes sensitized against the immunogenic preparation canbe done by techniques which are known to those who are skilled in theart. (See, for example, Douillard and Hoffinan, Basic Facts aboutHybridomas, in Compendium of Immunology, Vol. II, ed. by Schwartz, 1981;Kohler and Milstein, Nature 256:495-499, 1975; European Journal ofImmunology 6:511-519, 1976).

Unlike preparation of polyclonal antibodies, the choice of animal forthe isolation of sensitized lymphocytes is dependent on the availabilityof appropriate immortal lines capable of fusing with lymphocytes. Mouseand rat have been the animals of choice in hybridoma technology and arepreferably used. Humans can also be utilized as sources for sensitizedlymphocytes if appropriate immortalized human (or nonhuman) cell linesare available. For the purpose of the present invention, the animal ofchoice may be injected with an antigenic amount, for example, from about0.1 mg to about 20 mg of the protein or antigenic parts thereof. Usuallythe injecting material is emulsified in Freund's complete adjuvant.Boosting injections may also be required. The detection of antibodyproduction can be carried out by testing the antibody with appropriatelylabelled antigen. Lymphocytes can be obtained by removing the spleen oflymph nodes of sensitized animals in a sterile fashion and then used tocarry out fusion with the immortal cell line. Alternatively, lymphocytescan be stimulated or immunized in vitro, as described, for example, inReading, Journal of Immunological Methods 53: 261-291, 1982.

A number of cell lines suitable for fusion have been developed and thechoice of any particular line for hybridization protocols is directed byany one of a number of criteria such as speed, uniformity of growthcharacteristics, deficiency of its metabolism for a component of thegrowth medium, and potential for good fusion frequency.

Intraspecies hybrids, particularly between like strains, work betterthan interspecies fusions. Several cell lines are available, includingmutants selected for the loss of ability to secrete myelomaimmunoglobulin.

Cell fusion can be induced either by virus, such as Epstein-Barr orSendai virus, or polyethylene glycol. Polyethylene glycol (PEG) is themost efficacious agent for the fusion of mammalian somatic cells. PEGitself may be toxic for cells and various concentrations should betested for effects on viability before attempting fusion. The molecularweight range of PEG may be varied from 1000 to 6000. Best results areobtained when the PEG is diluted to from about 20% to about 70% (w/w) insaline or serum-free medium. Exposure to PEG at 37° C. for about 30seconds is preferred in the present case, utilizing murine cells.Extremes of temperature (i.e., about 45° C.) are avoided, andpreincubation of each component of the fusion system at 37° C. prior tofusion can be useful. The ratio between lymphocytes and malignant cellsis optimized to avoid cell fusion among spleen cells and a range of fromabout 1:1 to about 1:10 is commonly used.

The successfully fused cells can be separated from the myeloma line byany technique known by the art. The most common and preferred method isto choose a malignant line which is Hypoxthanine Guanine PhosphoribosylTransferase (HGPRT) deficient, which will not grow in anaminopterin-containing medium used to allow only growth of hybrids andwhich is generally composed of hypoxthanine 1×10⁻⁴M, aminopterin1×10⁻⁵M, and thymidine 3×10⁻⁵M, commonly known as the HAT medium. Thefusion mixture can be grown in the HAT-containing culture mediumimmediately after the fusion 24 hours later. The feeding schedulesusually entail maintenance in HAT medium for two weeks and then feedingwith either regular culture medium or hypoxthanine, thymidine-containingmedium.

The growing colonies are then tested for the presence of antibodies thatrecognize the antigenic preparation. Detection of hybridoma antibodiescan be performed using an assay where the antigen is bound to a solidsupport and allowed to react to hybridoma supernatants containingputative antibodies. The presence of antibodies may be detected by“sandwich” techniques using a variety of indicators. Most of the commonmethods are sufficiently sensitive for use in the range of antibodyconcentrations secreted during hybrid growth.

Cloning of hybrids can be carried out after 21-23 days of cell growth inselective medium (e.g., HAT). Cloning can be preformed by cell limitingdilution in fluid phase or by directly selecting single cells growing insemi-solid agarose. For limiting dilution, cell suspensions are dilutedserially to yield a statistical probability of having only one cell perwell. For the agarose technique, hybrids are seeded in a semi-solidupper layer, over a lower layer containing feeder cells. The coloniesfrom the upper layer may be picked up and eventually transferred towells.

Antibody-secreting hybrids can be grown in various tissue cultureflasks, yielding supernatants with variable concentrations ofantibodies. In order to obtain higher concentrations, hybrids may betransferred into animals to obtain inflammatory ascites.Antibody-containing ascites can be harvested 8-12 days afterintraperitoneal injection. The ascites contain a higher concentration ofantibodies but include both monoclonals and immunoglobulins from theinflammatory ascites. Antibody purification may then be achieved by, forexample, affinity chromatography.

Binding to the cell division protein contemplated herein may beaccomplished in a number of ways such as on a semi-solid growth mediumplate where colonies of bacteria are tested, by for example, the Westernblotting procedure. Alternatively, other semi-solid supports may beemployed onto which the sample containing the protein has beenimmobilized. In another method, the sample containing the protein iscontacted with a solid support already containing a specific antibody.In any event the principle behind the assay is the same. A wide range ofimmunoassay techniques are available as can be seen in U.S. Pat. Nos.4,016,043, 4,424,279 and 4,018,653, hereby incorporated by reference.This, of course, includes both single-site and two-site, or “sandwich”,assays of the non-competitive types, as well as in the traditionalcompetitive binding assays.

Sandwich assays are among the most useful and commonly used assays andare favored for use in the present invention. A number of variations ofthe sandwich assay technique exist, and all are intended to beencompassed by the present invention. Briefly, in a typical forwardassay, an unlabelled antibody is immobilized on a solid substrate andthe sample to be tested brought into contact with the bound molecule.After a suitable period of incubation, for a period of time sufficientto allow formation of an antibody-antigen secondary complex, a secondantibody specific to the antigen, labelled with a reporter moleculecapable of producing a detectable signal is then added and incubated,allowing time sufficient for the formation of a tertiary complex ofantibody-antigen-labelled antibody. Any unreacted material is washedaway, and the presence of the antigen is determined by observation of asignal produced by the reporter molecule. The results may either bequalitative, by simple observation of the visible signal, or may bequantitated by comparing with a control sample containing known amountsof hapten. Variations on the forward assay include a simultaneous assay,in which both sample and labelled antibody are added simultaneously tothe bound antibody. These techniques are well known to those skilled inthe art, including any minor variations as will be readily apparent.

In the typical forward sandwich assay, a first antibody havingspecificity for the enzyme or protein, or antigenic parts thereof,contemplated in this invention, is either covalently or passively boundto a solid surface. The solid surface is typically glass or a polymer,the most commonly used polymers being cellulose, polyacrylamide, nylon,polystyrene, polyvinyl chloride or polypropylene. The solid supports maybe in the form of tubes, beads, discs of microplates, or any othersurface suitable for conducting an immunoassay. The binding processesare well-known in the art and generally consist of cross-linkingcovalently binding or physically adsorbing, the polymer-antibody complexis washed in preparation for the test sample. An aliquot of the sampleto be tested is then added to the solid phase complex and incubated at25° C. for a period of time sufficient to allow binding of any subunitpresent in the antibody. The incubation period will vary but willgenerally be in the range of about 2-40 minutes. Following theincubation period, the antibody subunit solid phase is washed and driedand incubated with a second antibody specific for a portion of thehapten. The second antibody is linked to a reporter molecule which isused to indicate the binding of the second antibody to the hapten.

By “reporter molecule” as used in the present specification, is meant amolecule which, by its chemical nature, provides an analyticallyidentifiable signal which allows the detection of antigen-boundantibody. Detection may be either qualitative or quantitative. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores, luminescent molecules or radionuclide containingmolecules (i.e., radioisotopes).

In the case of an enzyme immunoassay, an enzyme is conjugated to thesecond antibody, generally by means of glutaraldehyde or periodate. Aswill be readily recognized, however, a wide variety of differentconjugation techniques exist, which are readily available to the skilledartisan. Commonly used enzymes include horseradish peroxidase, glucoseoxidase, beta-galactosidase and alkaline phosphatase, amongst others.The substrates to be used with the specific enzymes are generally chosenfor the production, upon hydrolysis by the corresponding enzyme, of adetectable color change. For example, p-nitrophenyl phosphate issuitable for use with alkaline phosphatase conjugates; for peroxidaseconjugates, 1,2-phenylenediamine, 5-aminosalicyclic acid, or toluidineare commonly used. It is also possible to employ fluorogenic substrates,which yield a fluorescent product rather than the chromogenic substratesnoted above. In all cases, the enzyme-labelled antibody is added to thefirst antibody hapten complex, allowed to bind, and then the excessreagent is washed away. A solution containing the appropriate substrateis then added to the tertiary complex of antibody-antigen-antibody. Thesubstrate will react with the enzyme linked to the second antibody,giving a qualitative visual signal, which may be further quantitated,usually spectrophotometrically, to give an indication of the amount ofhapten which was present in the sample. “Reporter molecule” also extendsto use of cell agglutination or inhibition of agglutination such as redblood cells on latex beads, and the like.

Alternately, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labelled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic color visually detectable with a lightmicroscope. As in the EIA, the fluorescent labelled antibody is allowedto bind to the first antibody-hapten complex. After washing off theunbound reagent, the remaining tertiary complex is then exposed to thelight of the appropriate wavelength the fluorescence observed indicatesthe presence of the hapten of interest. Immunofluorescent and EIAtechniques are both very well established in the art and areparticularly preferred for the present method. However, other reportermolecules, such as radioisotope, chemiluminescent or bioluminescentmolecules, may also be employed. It will be readily apparent to theskilled technician how to vary the procedure to suit the requiredpurpose.

Analogues And Homologues

The present invention contemplates the use of analogues, and inparticular ZipA and FtsZ protein analogues. Such analogues are definedas derivatives that have been modified structurally but that areobserved to function in an analogous manner [e.g., continue todemonstrate detectable binding, i.e., binding of approximately two fold(and more preferably approximately five fold) above the backgroundbinding of the negative control, under the conditions of the assaysdescribed herein (for example, using radiolabels and measuring countsper minute), albeit with different affinity than the parent(underivatized) molecule]. Those proteins that do not function in ananalogous manner are “non-ZipA” and/or “non-FtsZ” proteins. Proteinanalogues may be created by modification of the nucleotide sequenceencoding the zipA and/or ftsZ genes (e.g., by substitution, deletion,etc.). Alternatively, the present invention contemplates the use ofanalogues generated by synthesis of polypeptides in vitro such as bychemical means (or in vitro translation of mRNA).

The present invention also contemplates the use of homologues, which aredefined as the corresponding proteins for ZipA and FtsZ in otherspecies, including both Gram positive and Gram negative species. In oneembodiment, the present invention contemplates the use of homologousproteins from Haemophilus influenzae Rd, which is a small, nonmotile,Gram-negative bacterium whose only natural host is human. Six H.influenzae serotype strains (a through f) have been identified on thebasis of immunologically distinct capsular polysaccharide antigens.Non-typeable strains also exist and are distinguished by their lack ofdetectable capsular polysaccharide. They are commensal residents of theupper respiratory mucosa of children and adults and cause otitis mediaand respiratory tract infections, mostly in children. More seriousinvasive infection is caused almost exclusively by type b strains, withmeningitis producing neurological sequelae in up to 50% of affectedchildren.

The genome size of influenzae Rd is typical among bacteria. Morespecifically, the H. influenzae Rd genome is a circular chromosome of1,830,137 bp. The overall G+C nucleotide content is approximately 38%(A, 31%; C, 19%; G, 19%; T, 31%). The G+C-rich regions correspond to sixrRNA operons and a cryptic mu-like prophage. Genes for several proteinssimilar to proteins encoded by bacteriophage mu are located atapproximately position 1.56 to 1.59 Mbp of the genome. This area of thegenome has a markedly higher G+C content that average for H. influenzae(˜50% percent G+C compared to ˜38% for the genome overall).

The minimal origin of replication (oriC) in E. coli is a 245-bp regiondefined by three copies of a 13-bp repeat at one end (sites for initialDNA unwinding) and four copies of a 9-bp repeat at the other. Anapproximately 280-bp sequence containing structures similar to the three13-bp and four 9-bp repeats defines the putative origin of replicationin H. influenzae Rd. This region lies between sets of ribosomal operonsrrnF, rrnE, rrnD and rrnA, rrnB, rrnC. These two groups of ribosomaloperons are transcribed in opposite directions and the placement of theorigin is consistent with their polarity for transcription.

Termination of E. coli replication is marked by two 23-bp terminationsequences located ˜100 kb on either side of the midway point at whichthe two replication forks meet. Two potential termination sequencessharing a 10-bp core sequence with the E. coli termination sequence havebeen identified in H. influenzae. These two regions are offsetapproximately 100 kb from a point approximately 180° opposite of theproposed origin of H. influenzae replication.

A homologue gene encoding ZipA in H. influenzae is located (as with E.coli) adjacent to the DNA ligase gene. FIG. 5 shows the amino acidsequence of the ZipA protein from E.coli (top sequence) aligned with thehomologue from H. influenzae (bottom sequence) (SEQ ID NO:3). Thealignment shown in FIG. 5 was generated using the Bestfit proghram(BLAST Network Servie, National Center for Biotechnology Information).Gaps were introduced to provide maximum alignment between the twosequences (dots within a strand indicate a gap). Vertical lines betweenthe two sequences indicate regions of identity. A double dot indicates aconservative substitution, while a single dot between the strandsindicates a less conservative substitution.

There are a number of regions of striking homology, including but notlimited to: ILIIVG (SEQ ID NO:4); DLX(R or N)X(L or T)ILIIVG (SEQ IDNO:5); ILIIVGX(Aor I)X(I or V)AX(I or L)X(I or V)AL (SEQ ID NO:6);ILIIVGX(A or I)X(I or V)AX(I or L)X(I or V)ALX(I or L)VHG (SEQ ID NO:7);ALX(I or L)VHG (SEQ ID NO:8); ALX(I or L)VHGX(F or L)W (SEQ ID NO:9);YHRHL (SEQ ID NO:10); PX(A or V)LFSX(L or V)AN (SEQ ID NO:11); PGTF (SEQID NO:12); X(I or L)FMQ (SEQ ID NO:13); X(I or L)FMQX(V or L)PS (SEQ IDNO:14). The present invention contemplates ZipA protein homologueshaving one or more of these regions of homology.

DETAILED DESCRIPTION OF THE INVENTION

The detailed description of the invention is divided into five majorsections: I) Gene Isolation and Plasmid Construction; II) ProteinExpression and Purification; III) Protein Labelling; IV) Binding ToOther Proteins; and V) Antimicrobial Screening.

I. Gene Isolation And Plasmid Construction

Isolation of the E. coli zipA gene and construction of plasmidscontaining the zipA gene was performed as generally described bySambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., (ColdSpring Harbor Press, NY 1989). The zipA gene was isolated by expressioncloning [schematically represented in FIG. 6; FIG. 6 shows that thelabelled FtsZ fusion protein can be used to probe an expression libraryto identify recombinant clones expressing proteins which bind to FtsZ(“Expression Library ”)as well as to probe Western blots to identifyprotein species separated by electrophoresis which bind to FtsZ (“FarWestern”)]. Briefly, a λgt11 library was made from chromosomal DNA of E.coli strain PB103. Plaques were lifted to nitrocellulose filters andprobed with ³²P-labelled HKFT-FtsZ to identify clones capable ofexpressing ZipA (the expression, purification and labelling of HKFT-FtsZis described below). In total 7 recombinant phage were identified, allof which contained 7-10 Kb of E. coli DNA, including the complete zipAgene. The nucleotide sequence of the zipA gene is listed in SEQ ID NO:1and the amino acid sequence of the ZipA protein is listed in SEQ IDNO:2.

FIG. 7 shows a portion of the E. coli genome containing the zipA genewhich was present on the recombinant phage clones. As shown at the topof FIG. 7, the zipA gene is located between the cysZ gene and the liggene, the latter encoding DNA ligase. In FIG. 7, the followingabbreviations are used: B (BamHI), Pv (PvuI), Ag (AgeI), P (PstI), Af(AflII), H (HindIII). The large P under an arrow indicates the locationof the promoter driving the expressing of either the cysZ, zipA or liggenes; the direction of the arrowhead indicates the direction oftranscription. The DNA sequence found between the BamHI and HindIllsites shown at the top of FIG. 7 which contains the complete zipA geneas well as portions of the cysZ and lig genes is listed in SEQ ID NO:1and shown in FIG. 1. A number of plasmid constructs containing zipAsequences are shown schematically in FIG. 7 (only sequences containingzipA sequences are depicted; i.e., the plasmid backbone is notdepicted). pDB315 contains a 1727 bp BamHI-HindIII fragment containingthe entire zipA gene and portions of the cysZ and lig genes; thisBamHI-HindIII fragment was isolated from one of the seven λgt11 clones.The generation of pDB315 and the remaining plasmids is discussed below.

To permit over-expression of the ZipA protein in bacterial cells, thecomplete zipA ORF was inserted into the pET21b vector which allowed theexpression of ZipA under the control of the T7 promoter; the resultingplasmid was termed pDB319. The construction of pDB319 and a number ofplasmids which encode fragments of the ZipA protein is describe below.In addition, the construction of vectors encoding fusion proteins whichare employed in the screening methods of the present invention are alsodescribed.

a) Construction Of Vectors Encoding The ZipA Protein And FragmentsThereof

Plasmids pDB319 and pDB322 express the complete zipA gene under thetranscriptional control of the T7 or lac promoter, respectively. Toconstruct pDB319 and pDB322, zipA was amplified by a PCR with primers5′-ACAGAGATCCATATGATGCAGGATTTGCGTCTG-3′ (SEQ ID NO:15) and5′-TTAACCAAGCTTAAGTGTATCAGGCGTTGG-3′ (SEQ ID NO:16) designed tointroduce a Ndel site at the translation start codon (underlined) ofzipA. Chromosomal DNA isolated from E. coli strain PB103 was used astemplate. The 1018 bp PCR product was treated with HindIII and NdeI, andthe resulting 986 bp fragment was ligated to pET21a (Novagen) which hadbeen digested with HindIII and NdeI to generate pDB317.

The 676 bp Agel-Hindlll fragment of pDB317 was then replaced with the1015 bp Agel-HindIII fragment of pDB315 (described below), yieldingpDB318. To obtain pDB319, pDB318 was treated successively with AflII andHindIII, Klenow enzyme plus deoxynuceotides, and ligase, therebyremoving all lig sequences and retaining a HindIII site. Plasmid pDB319encodes the complete ZipA protein under the control of the T7 promotor.To place zipA expression under control of the lac promotor, the 1089BglII-HindIII fragment of pDB319 was ligated to BamHI-Hindlll-digestedpMLB1113, yielding pDB322 [the pMLB plasmids are derived from pBR322 andcarry a polylinker region flanked on one side by the lacl gene, the lacpromotor and operator, and the lacZ ribosome binding site andtranslation start codon, and on the other side by the rest of the lacZgene and part of lacy; the plasmids also contain the beta-lactamase genefor ampicillin resistance].

Plasmids encoding fragments of ZipA were constructed as follows. ForpDB315 and pDB316, the 1727 bp BamHI-HindIII fragment from the λgt11derivative λCH1-1A (zipA⁺) (i.e., one the original λgt11 clones) wasfirst inserted in M13 mp19 (Phannacia), yielding M13 mp19H-B1.7. A 338bp deletion within zipA was created by digestion of M13 mp19H-B1.7 DNAwith PstI, and recirculization of the large fragment, resulting in M13mp19H-B1.7ΔPstI. The 1727 bp and 1389 bp BamHI-HindIII fragments fromthe two phages were next cloned into pMAK700 [this vector was chosen tobe able to introduce a knock-out of the zipA gene in the chromosome;pMAK700 is a pSC101 derivative but contains a repAts allele whichrenders the plasmid temperature sensitive for replication, as well as achloramphenicol resistance marker], yielding pDB315 (zipA⁺) [whichcontains the complete zipA gene] and pDB316 (zipA⁻) [which contains thesame fragment but with a 330 bp PstI fragment, internal to zipA, deleted(thus zipA−)], respectively.

b) Construction Of Plasmids Encoding ZipA/GFP Fusion Proteins

The present invention contemplates “fusion labelling” of the proteinsuseful in the screening method. In one embodiment, the present inventioncontemplates a fusion protein comprising ZipA (or a portion thereof) andthe green fluorescent protein (GFP) from the jellyfish Aequoreavictoria. GFP emits green light (approximately 510 nm).

Unlike other bioluminescent reporters, GFP fluoresces in the absence ofany other proteins, substrates, or cofactors.

Purified GFP is a 27-kDa monomer consisting of 238 amino acids. Whileintact GFP is required for fluorescence, the active chromophore is ahexapeptide which contains a cyclized Ser-dehydroTyr-Gly trimer.Chromophore formation is oxygen-dependent and occurs gradually aftertranslation.

GFβ-containing vectors are commercially available from ClontechLaboratories, Inc. (Palo Alto, Calif.) (hereinafter “Clontech”). pGFP(Clontech) is primarily intended as a source of the GFP cDNA, which canbe readily excised using restriction enzyme sites in the two multiplecloning sequences (MCS) flanking the gfp coding sequences.Alternatively, the GFP coding sequences can simply be amplified from anyplasmid containing the GFP gene by PCR. pGFP-1 (Clontech) is a versatiletranscription reporter vector for monitoring the activity of promoterscloned into the MCS upstream of the promoterless GFP gene. The sequencesaround the GFP start codon have been converted to a Kozak consensustranslation initiation site to increase translation efficiency ineukaryotic cells. The vector also contains aneomycin/kanamycin-resistance cassette for selection for selection oftransformed bacterial and eukaryotic cells. pGFβ-N1 is one of threevectors which are useful for fusing heterologous proteins to theN-terminus of GFP. The same MCS is present in a different reading framein pGFβ-N1, pGFβ-N2, and pGFβ-N3. These constructs enable the use of anyone of 17 restriction sites to create an in-frame fusion to a convenientrestriction site in the gene of interest. Similarly, pGFβ-C1, pGFβ-C2,and pGFβ-C3 (Clontech) can be used to create in-frame fusions to theC-terminus of GFP. All six GFP protein fusion vectors contain the Kozakconsensus sequence, the immediate early promoter of cytomegalovirus(CMV) to express fusions in mammalian cells, and aneomycin/kanamycin-resistance cassette.

Plasmids pDB341 and pCH50 (shown schematically in FIG. 7) encodeZipA/GFP fusion proteins. For pDB341 and pCH50, zipA was amplified by aPCR as above, except that the downstream primer used was5′-AAGTCTCGAGGGCGTTGGCGTCTTTGAC-3′ (SEQ ID NO:17). This primer wasdesigned to substitute the translation stopcodon with an XhoI site(underlined). The PCR product was treated with NdeI and XhoI, and the984 bp fragment ligated to NdeI and XhoI-digested pET21b, resulting inpCH38. Plasmid pGFPS65T is a mutant derived from pGFP by substituting aserine at position 65 for a threonine; the plasmid contains gfpS65T on a729 bp BamHI fragment in the vector pRSET B (available commercially fromInvitrogen, Madison, Wis.). This fragment was ligated to BamHI-digestedpET16b yielding plasmid pDB338 which contains an XhoI site immediatelyupstream of the gfp coding sequence. The small ApaI-XhoI fragment ofpDB338 was next replaced by that of pCH38, resulting inpDB341[PT7::ZipA-GfpS65T]. This plasmid encodes a 64.2 kd ZipA-GfpS65Tfusion protein which includes the complete ZipA and Gfp proteins, fusedby a small linker peptide (LEDPPAEF) (SEQ ID NO:18). To place expressionof this fusion under control of the lac promotor, the ˜2180 bpBglII-HindIII fragment of pDB341 was next inserted into the BamHI andHindIII sites of pMLB1113, yielding pCH50 [P_(lac)::ZipA-GfpS65T].

c) Construction Of Plasmids Encoding MinE And FtsZ Fusion Proteins

Plasmids encoding fusion proteins comprising the MinE and FtsZ proteinswere constructed to allow the over-expression of these proteins used toprovide negative and positive controls respectively in the methods ofthe present invention. As the MinE protein does not interact with theZipA protein, it is useful as a negative control. In contrast, the FtsZprotein interacts with the ZipA protein, and it is useful as a positivecontrol.

i) Plasmids Encoding MinE Fusion Proteins

pDB311 encodes a 15.9 kD HFKT-MinE fusion protein, in which theN-terminal four amino acids of MinE are replaced with the HFKT peptideMGHHHHHHHHHHSSGHIEGRHMDYKDDDDKARRASVEFHMASMTGGQQMGRGS (SEQ ID NO: 19),under control of the T7 promoter. To construct plasmid pDB311, pDB 151was treated successively with TaqI, Klenow enzyme plus deoxynucleotides,and EcoRI. The 307 bp fragment, coding for all but the first four aminoacids of MinE, was ligated to pET21a (Novagen) that had been treatedsuccessively with BamHI, Klenow enzyme plus deoxynucleotides, and EcoRI.This yielded pDB302, in which the BamHI site was restored as expected,but the EcoRI site was fortuitously destroyed. Plasmid PDB302 encodes aT7.tag-MinE (T-MinE) fusion protein, in which the N-terminal four aminoacids of MinE are replaced with the T7.tag peptide MASMTGGQQMGRGS (SEQID NO:20), under control of the T7 promoter of the vector.

The 1037 bp ApaI-NdeI fragment of pDB302 was replaced with the 1099 bpApaI-NdeI fragment of pET16b (Novagen). The resulting plasmid, pDR1,encodes a fusion protein, HT-MinE in which the His-tag peptideMGHHHHHHHHHHSSGHIEGRH (SEQ ID NO:21) is fused to the N-terminus of theT-MinE protein. Plasmid pAR(ΔRI)59/60 [see generally, M. A. Blanar andW. J. Rutter, “Interaction Cloning: Identification of a helix-loop-helixZipper Protein that Interacts with cFos,” Science 256:1014 (1992)]contains a 54 bp NdeI fragment encoding the peptide MDYKDDDDKARRASVEF(SEQ ID NO:22). This peptide, denoted as FK, is a fusion of the Flagpeptide (IBI) with a Heart Muscle Kinase substrate peptide (underlined).To ease the manipulation of the NdeI cassette, a ˜1800 bp EcoRIfragment, isolated from pUC4-KIXX (Pharmacia) and carrying the Tn5aph(neo) gene, was inserted in the EcoRI site of pAR(ΔRI)59/60 which isflanked by the NdeI sites. The ˜1850 bp NdeI fragment of the resultingplasmid, pTD1, was next inserted in the NdeI site of pDR1, yieldingpDB309. Finally, pDB311 was obtained by deletion of the ˜1800 bp Tn5stuffer-fragment by treatment with EcoRI and re-ligation.

ii) Plasmids Encoding FtsZ Fusion Proteins

Plasmid pDR10 encodes a 46.6 kD HFKT-FtsZ fusion protein in which thepeptide MGHHHHHHHHHHSSGHIEGRHMDYKDDDDKARRASVEFHMASMTGGQQMGRGSH (SEQ IDNO:23) is fused to the complete FtsZ protein.

To construct plasmid pDR10 chromosomal DNA from E. coli strain PB103[see generally, P. A. J. de Boer et al., “Isolation and Properties ofminb, a Complex Genetic Locus Involved in Correct Placement of theDivision Site in Escherichia coli” J. Bacteriol. 170:2106 (1988)] wasused as a template in a PCR to amplify ftsZ. Primers[5′-GGAGGATCCCATATGTTTGAACCAATGGAAC-3′ (SEQ ID NO:24) and5′-TTCCGGTCGACTCTTAATCAGCTTGCTTACG-3′ (SEQ ID NO:25)] were designed tointroduce a BamHI site near the translation start codon (underlined) offtsZ. The resulting 1176 bp PCR product was treated with BamHI and SalIto yield a 1163 bp fragment which was used to replace the 320 bpBamHI-SalI fragment, containing all minE sequences, from pDB311. Theresulting plasmid was termed pDR10.

II. Protein Expression and Purification

MinE and FtsZ fusion proteins (both containing the HFKT peptide) wereexpressed in E. coli and purified as follows. Strain HMS 174(DE3)/pLysS(Novagen) was transformed with either pDR10 or pDB311. HMS174(DE3)/pLysScontaining either pDR10[PT7::hfkt-ftsZ] or pDB311[PT7::hfkt-minE] weregrown shaking overnight at 37° C. in LB medium with ampicillin (100μg/ml), chloramphenicol (25 μg/ml) and glucose (0.2%). Cultures werediluted 200× in 500 ml of LB with 50 μg/ml ampicillin and 0.04% glucose.At an OD₆₀₀ of approximately 0.5, IPTG (isopropyl-β-thiogalactoside) wasadded to 0.84 mM, and growth was continued for 90 min. Cells werecollected by centrifugation (5000×g) and washed once in 20 ml of coldsaline 0.9% NaCl. Pellets were quickly frozen in a dry-ice/acetone bathand stored at −85° C. Cells were quickly thawed at 37° C., andresuspended in 5.0 ml of cold buffer A (20 mM Tris.Cl, 70 mM NaCl, 50 mMimidazole, pH 7.9). Cell lysis was induced by three additional cycles ofrapid freezing (dry-ice/acetone bath) and thawing (37° C. waterbath).

The lysate was briefly sonicated to reduce viscosity, and insolublematerial was removed by centrifugation at 200,000×g for 3 hr at 8° C. Aportion of the supernatant (50 mg total protein) was passed 3 times overa 0.5 ml Fast Flow Chelating Sepharose (Pharmacia) column, which hadpreviously been charged with NiSO₄ and equilibrated in buffer A. Thecolumn was washed 3 times with 1.5 ml of buffer B (20 mM Tris-Cl, 500 mMNaCl, 50 mM imidazole, pH 7.9), and 3 times with 1.0 ml of buffer C (20mM Tris-Cl, 500 mM NaCl, 200 mM imidazole, pH 7.9). The HFKT-taggedprotein was then eluted in 3×1.0 ml of buffer D (20 mM Tris-Cl, 500 mMNaCl, 500 mM imidazole, pH 7.9). Appropriate fractions (determined bySDS PAGE) were pooled, dialyzed extensively against buffer E (20 mMTris-Cl, 25 mM NaCl, 2 mM EDTA, pH 8.0), concentrated in a Centricon 10device (Amicon), and stored frozen at −85° C. The HFTK-tagged proteinswere estimated to be greater than 95% pure as judged by SDS-PAGE andCoomassie Brilliant Blue staining (approximately 25 micrograms ofprotein were loaded on a 12% acrylamide gel).

III. Protein Labelling

The purified HFKT-tagged proteins were phosphorylated in vitro byincubation with [γ³²P]ATP and the catalytic subunit of bovine heartmuscle kinase (Sigma Chemical Co., St. Louis, Mo.). Reactions (30 μl)were performed on ice for 45 min, and contained HMK-buffer (20 mMTris-Cl, 100 mM NaCl, 12 mM MgCl₂, 1 mM DTT, pH 7.5), 300-600 pmol (9-15μg) purified HFKT-tagged protein, 60 μCi [γ-³²P]ATP (6000 Ci/mmol), and1 μl of kinase (10 U/μl in 40 mM DTT). To separate protein from otherreaction components, 20 μl of buffer Z (25 mM HEPES-KOH, 100 mM KCL, 12mM MgCl₂, 1 mM DTT, 10% glycerol, pH 7.7) was added, and the mixture wasloaded onto a 2.0 ml Kwiksep Excellulose desalting column (Pierce),which had previously been washed with 5.0 ml of buffer Z containing 1mg/ml BSA (bovine serum albumin), and 5.0 ml of buffer Z without BSArespectively. The excluded volume was collected in fractions of 50 μl,and aliquots were used to determine radioactivity by scintillationcounting. To visualize the presence and integrity of the desiredradiolabeled species, aliquots were electrophoresed on SDS-PAGE gelsfollowed by autoradiography of dried gels. Peak fractions were pooledand kept on ice until further use. Typical specific activities obtainedwere 1.5 to 4.0×10⁶ cpm/μg HFKT-MinE and 5.0 to 8.0×10⁶ cpm/μgHFKT-FtsZ.

IV. Use of Immoblized Fusion Proteins in Binding Assays

Affinity Blotting

After the addition of one volume of electrophoresis sample buffer (125mM Tris.Cl, 4% SDS, 20% glycerol, 1.4 M β-mercaptoethanol, pH=6.6),samples (e.g, whole cells boiled in sample buffer, or fractionatedcells) were held in a boiling water bath for 5 min., and subjected toconventional SDS-PAGE on 0.75 mm thick 10%T/2.7%C gels. Electrophoretictransfer to nitrocellulose filter (0.2 μ) was performed in a Genieblotter (Idea Scientific Co.) with regular Towbin buffer (25 mM Tris.Cl,192 mM glycine, 20% methanol, pH 8.3) at 12 V for 30 min. To monitor thetransfer and to visualize the molecular weight standards, filters weretreated with the reversible stain Ponceau S (0.1% in 5% acetic acid) for1 min. Filters were next destained in 10 mM Tris-Cl, rinsed in water,and allowed to dry for 15 min in air. Filters were wetted in HBB buffer(25 mM HEPES-KOH, 25 mM NaCl, 5 mM MgCl₂, 1 mM DTT, pH 7.7), and blockedfor 60 min in HBB with 5% non-fat dried milk and 0.05% Np-40, and for 30min in HBB with 1% non-fat dried milk and 0.02% Np-40. They were nextincubated overnight with labelled protein at 275,000 cpm/ml in Hybbuffer (20 mM HEPES-KOH, 50 mM KCL, 0.1 mM EDTA, 2.5 mM MgCl₂, 0.1 mMATP, 1% non-fat dried milk, 0.02% Np-40, 1 mM DTT, pH 7.7), and washedthree times for 7, 5, and 2 min, respectively, in Hyb buffer withoutlabelled protein. All blocking, incubation, and washing steps wereperformed at 4° C. Filters were air dried and analyzed by exposing X-rayfilm, or by quantitatively imaging of radioactivity on an Ambisphosphorimager.

Membrane Localization of ZipA

For detergent solubilization of membrane proteins, an overnight cultureof strain PB103 was diluted 200× in 1 liter of LB medium, and incubateduntil an OD₆₀₀ of 1.0 was reached. Cells were collected bycentrifugation, washed once in cold saline, resuspended in 9.0 ml ofcold cell breaking buffer (CBB conatins 20 mM Tris-Cl, 25 mM NaCl, 5 mMEDTA, 3.6 mM β-mercaptoethanol, pH 8.0), and broken by three passagesthrough a French pressure cell followed by very brief sonication toreduce viscosity. The pressate was subjected to centrifugation at200,000×g for 3 hrs at 8° C., the supernatant (S200) removed, and thepellet fraction (P200) resuspended in 2.0 ml of CBB. Aliquots (0.1 ml,5.4 mg protein) of this were brought up to 0.5 ml of either CBB alone,or CBB with 6 M urea, 0.5% Triton-X100, or 0.2% Sarkosyl (finalconcentrations), and the mixtures were incubated at room temperature ina head over head mixer for 1 hr. Soluble material was separated frominsoluble material by centrifugation (200,000×g for 1 hour at 4 C.), thelatter homogenized in 0.5 ml CBB by sonication (setting 2, approximately20 seconds, on ice), and equal samples of each used for SDS-PAGE andaffinity blotting.

To fractionate crude membrane preparations into the different membranecomponents, the procedure of Ishidate et al. was modified as follows.Briefly, cells from a logaritmically growing culture (500 ml LB) ofstrain PB103 were harvested at an OD₆₀₀ of 1.0 by centrifugation, washedwith cold saline, and resuspended in 10 ml of 20% sucrose (All sucrosesolutions were prepared w/w, in 10 mM Hepes-KOH, pH 7.4) and 10 μg/mleach of DNase and RNase. Cells were broken by three passages through aFrench pressure cell (10,000 psi) after which EDTA was added to 5 mM. Toobtain a crude membrane preparation, 1.7 ml of the pressate was loadedon each of two gradients (SG0), which were prepared by layering 2.5 ml25% sucrose on top of an 0.8 ml 60% sucrose cushion. The gradients werespun in an SW50.1 rotor at 37,000×g for 4 hr at 4° C. Crude membrane wasrecovered from the cushion by aid of a syringe, diluted with 10 mMHEPES-KOH (pH 7.4) to a refractive index of 1.3650 and a volume of 1.7ml, and loaded on top of a sedimentation sucrose gradient (SGI) preparedby layering 60% (0.5 ml), 55% (1.0 ml), 50% (2.1 ml), 45% (2.1 ml), 40%(2.1 ml), 35% (1.5 ml), and 30% (1.0 ml) sucrose solutions. Aftercentrifugation in an SW41 rotor at 39,000×g for 20 hrs at 4° C.,fractions (0.33 ml) were collected from the bottom of the tube. Theprotein concentration and refractive index were determined for eachfraction, and the latter was converted to the corresponding specificgravity value using ISCO tables (9th edition). Appropriate fractionswere pooled and each pool was adjusted to a volume of 2.0 ml and arefractive index of 1.4285, by addition of 10 mM HEPES-KOH (pH 7.4) andpowdered sucrose. From each pool, 0.2 ml was saved, and the rest wasincorporated into a floatation sucrose gradient (SG2) which consisted,from bottom to top, of 67% (0.5 ml), pooled sample (1.8 ml), 50% (1.8ml), 45% (3.0 ml), 40% (2.0 ml), 35% (1.0 ml), and 30% sucrose. Aftercentrifugation in an SW41 rotor at 36,000×g for 72 hrs at 4° C.,fractions (0.30 ml) were collected and analyzed as above, after whichappropriate fractions were pooled. These pooled fractions, as well asthe remainder of the pooled fractions from SG1, were adjusted to asucrose concentration of less than 10% by addition of CBB to a volume of10 ml. Membranes were colected by centrifugation in an SW50.1 rotor at38,000×g for 2 hours at 4 C., and resuspended in 0.1 ml CBB. Aliquotswere subsequently subjected to SDS-PAGE and affinity blotting asdescribed above.

The results (not shown) can be summarized as follows. First, ZipAco-fractionated with the inner membrane fraction of cells during bothsedimentation and floatation in sucrose gradients. This demonstratesthat ZipA is truly associated with the inner membrane. Second, ZipAcould be solubilized by detergent but not by urea. This indicates thatZipA is an integral membrane protein (i.e., traverses the membrane).

Yeast Two-hybrid System

While the present invention allows for a cell-free screening assay,cellular screening assays are also contemplated. A Yeast two-hybridsystem (commercially available from Clontech) allows for the detectionof protein—protein interactions in yeast. See generally, Ausubel et al.,Current Protocols in Molecular Biology (John Wileyt & Sons)(pp.13.14.1-13.14.14). The system can be used to screen speciallyconstructed cDNA libraries for proteins that interact with a targetprotein (e.g., ZipA or FtsZ proteins or fragments thereof). The presentinvention contemplates the use of the two-hybrid system to screen forcompounds that will bind to either the ZipA protein or FtsZ protein.Compounds (e.g., proteins) identified in a two-hybrid creen which bindto the FtsZ protein and which are not ZipA proteins may representcompounds capable of blocking the interaction of ZipA protein and FtsZprotein. Similarly, compounds which bind to the ZipA protein which arenot FtsZ proteins may represent compounds capable of blocking theinteraction of ZipA and FtsZ.

From the above, it should be clear that the present invention providescompounds and methods for screening large numbers of test compounds forantimicrobial activity. By using bacterial proteins in vitro to detectcompounds, the present invention allows for a cell-free screeningsystem.

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 25(2) INFORMATION FOR SEQ ID NO: 1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 2160 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #1:CAATACCAGG GATGAAGTAA AGAATTAGTA ATACAATTGC GCGCGGCAGA TA#CCAGGCAA     60ATTTTTGCCA TTCGCGTTTC ATGATTCGCG GCACATCTTT CATGATACCG AA#AATCCCGG    120TATCTGGCGG TGTAGCGCCA GTCAATCGTG CTTCCAGTTG TTCAGCCAAT AA#ACCGTTAA    180ACGGAGCGGC AATCCAGTTA GCAATCGTGG AGAAGAAATA GCCAAACACT AA#CAGCACAG    240AGATGACACG CAGAGGCCAC AACAGATAAC TCAGCCATTG TAGCCAGTCC GG#AACGTAAC    300TCATGAGAGT CGGGATCCAG ACATCGAGCT GTGTAAAGAG CCACCAGAAT GC#GCCCCCCA    360TCAACAAAAT ATTGACCAGC AGCGGTAAAA TAACGAAACG CCGAATCCCA GG#TTGCGAGA    420CGAGCTTCCA GCCTTGCGCA AAATAGTAAA AACCGCTGCG TGGGGCAGAT GT#GAATGATG    480AAACCATAAT CAGGATGAGC TCCTTTTGAC CAATCCCAGG AAAATTCTGC GT#ATTTTACC    540GGGTAATTGC GCAATGGACA GTTAGGATAT GTTCGAAAAA ACAGCAAAAA GC#ACGATTTC    600ATCTATCTTT GTGCTGTGAA AGTTAATAGT GCACTTGCAC TTGAGGTAAT CG#GCAAATAC    660TCTTAGTGAG TAAATGTTTG CCGTGGTGGC AAGGTGTTAG AACAACAGAG AA#TATAATGA    720TGCAGGATTT GCGTCTGATA TTAATCATTG TTGGCGCGAT CGCCATAATC GC#TTTACTGG    780TACATGGTTT CTGGACCAGC CGTAAAGAAC GATCTTCTAT GTTCCGCGAT CG#GCCATTAA    840AACGAATGAA GTCAAAACGT GACGACGATT CTTATGACGA GGATGTCGAA GA#TGATGAGG    900GCGTTGGTGA GGTTCGTGTT CACCGCGTGA ATCATGCCCC GGCTAACGCT CA#GGAGCATG    960AGGCTGCTCG TCCGTCGCCG CAACACCAGT ACCAACCGCC TTATGCGTCT GC#GCAGCCGC   1020GTCAACCGGT CCAGCAGCCG CCTGAAGCGC AGGTACCGCC GCAACATGCT CC#GCATCCAG   1080CGCAGCCGGT GCAGCAGCCT GCCTATCAGC CGCAGCCTGA ACAGCCGTTG CA#GCAGCCAG   1140TTTCGCCACA GGTCGCGCCA GCGCCGCAGC CTGTGCATTC AGCACCGCAA CC#GGCACAAC   1200AGGCTTTCCA GCCTGCAGAA CCCGTAGCGG CACCACAGCC TGAGCCTGTA GC#GGAACCTG   1260CTCCAGTTAT GGATAAACCG AAGCGCAAAG AAGCGGTGAT TATCATGAAC GT#CGCGGCGC   1320ATCACGGTAG CGAGCTAAAC GGTGAAGCTC TTCTTAACAG CATTCAACAA GC#GGGCTTCA   1380TTTTTGGCGA TATGAATATT TACCATCGTC ATCTTAGCCC GGATGGCAGC GG#CCCGGCGT   1440TATTCAGCCT GGCGAATATG GTGAAACCGG GAACCTTTGA TCCTGAAATG AA#GGATTTCA   1500CTACTCCGGG TGTCACTATC TTTATGCAGG TACCGTCTTA CGGTGACGAG CT#GCAGAACT   1560TCAAGCTGAT GCTGCAATCT GCGCAGCATA TTGCCGATGA AGTGGGCGGT GT#CGTGCTTG   1620ACGATCAGCG CCGTATGATG ACTCCGCAGA AATTGCGCGA GTACCAGGAC AT#CATCCGCG   1680AAGTCAAAGA CGCCAACGCC TGATACACTT AAGGCAAATT AACTCCTCTT CG#AACCCCCG   1740CTTGTCGGGG GTTTTTAGCA TTGATGGTGC GATATGGAAT CAATCGAACA AC#AACTGACA   1800GAACTGCGAA CGACGCTTCG CCATCATGAA TATCTTTATC ATGTGATGGA TG#CGCCGGAA   1860ATTCCCGACG CTGAATACGA CAGGCTGATG CGCGAACTGC GCGAGCTGGA AA#CCAAACAT   1920CCAGAACTGA TTACGCCTGA TTCGCCTACT CAACGTGTAG GCGCTGCGCC GC#TGGCGGCT   1980TTCAGCCAGA TACGCCATGA AGTACCAATG CTGTCACTGG ATAACGTTTT TG#ATGAAGAA   2040AGCTTTCTTG CTTTCAACAA ACGTGTGCAG GACCGTCTGA AAAACAACGA GA#AAGTCACC   2100TGGTGCTGTG AGCTGAAGCT GGATGGTCTT GCCGTCAGTA TTCTGTATGA AA#ATGGCGTT   2160 (2) INFORMATION FOR SEQ ID NO: 2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 328 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: Not R#elevant           (D) TOPOLOGY: Not Relev #ant    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #2:Met Met Gln Asp Leu Arg Leu Ile Leu Ile Il #e Val Gly Ala Ile Ala1               5    #                10   #                15Ile Ile Ala Leu Leu Val His Gly Phe Trp Th #r Ser Arg Lys Glu Arg            20       #            25       #            30Ser Ser Met Phe Arg Asp Arg Pro Leu Lys Ar #g Met Lys Ser Lys Arg        35           #        40           #        45Asp Asp Asp Ser Tyr Asp Glu Asp Val Glu As #p Asp Glu Gly Val Gly    50               #    55               #    60Glu Val Arg Val His Arg Val Asn His Ala Pr #o Ala Asn Ala Gln Glu65                   #70                   #75                   #80His Glu Ala Ala Arg Pro Ser Pro Gln His Gl #n Tyr Gln Pro Pro Tyr                85   #                90   #                95Ala Ser Ala Gln Pro Arg Gln Pro Val Gln Gl #n Pro Pro Glu Ala Gln            100       #           105       #           110Val Pro Pro Gln His Ala Pro His Pro Ala Gl #n Pro Val Gln Gln Pro        115           #       120           #       125Ala Tyr Gln Pro Gln Pro Glu Gln Pro Leu Gl #n Gln Pro Val Ser Pro    130               #   135               #   140Gln Val Ala Pro Ala Pro Gln Pro Val His Se #r Ala Pro Gln Pro Ala145                 1 #50                 1 #55                 1 #60Gln Gln Ala Phe Gln Pro Ala Glu Pro Val Al #a Ala Pro Gln Pro Glu                165   #               170   #               175Pro Val Ala Glu Pro Ala Pro Val Met Asp Ly #s Pro Lys Arg Lys Glu            180       #           185       #           190Ala Val Ile Ile Met Asn Val Ala Ala His Hi #s Gly Ser Glu Leu Asn        195           #       200           #       205Gly Glu Ala Leu Leu Asn Ser Ile Gln Gln Al #a Gly Phe Ile Phe Gly    210               #   215               #   220Asp Met Asn Ile Tyr His Arg His Leu Ser Pr #o Asp Gly Ser Gly Pro225                 2 #30                 2 #35                 2 #40Ala Leu Phe Ser Leu Ala Asn Met Val Lys Pr #o Gly Thr Phe Asp Pro                245   #               250   #               255Glu Met Lys Asp Phe Thr Thr Pro Gly Val Th #r Ile Phe Met Gln Val            260       #           265       #           270Pro Ser Tyr Gly Asp Glu Leu Gln Asn Phe Ly #s Leu Met Leu Gln Ser        275           #       280           #       285Ala Gln His Ile Ala Asp Glu Val Gly Gly Va #l Val Leu Asp Asp Gln    290               #   295               #   300Arg Arg Met Met Thr Pro Gln Lys Leu Arg Gl #u Tyr Gln Asp Ile Ile305                 3 #10                 3 #15                 3 #20Arg Glu Val Lys Asp Ala Asn Ala                 325(2) INFORMATION FOR SEQ ID NO: 3:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 327 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: Not R #elevant          (D) TOPOLOGY: Not Relev #ant     (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #3:Asp Leu Asn Thr Ile Leu Ile Ile Val Gly Il #e Val Ala Leu Val Ala1               5    #                10   #                15Leu Ile Val His Gly Leu Trp Ser Asn Arg Ar #g Glu Lys Ser Lys Tyr            20       #            25       #            30Phe Asp Lys Ala Asn Lys Phe Asp Arg Thr Se #r Leu Thr Ser Arg Ser        35           #        40           #        45His Thr Gln Glu Glu Met Val Gln Pro Asn As #n Ile Ser Pro Asn Thr    50               #    55               #    60Tyr Val Glu Asn Gly His Thr Pro Ile Pro Gl #n Pro Thr Thr Glu Lys65                   #70                   #75                   #80Leu Pro Ser Glu Ala Glu Leu Ile Asp Tyr Ar #g Gln Ser Asp Lys Ser                85   #                90   #                95Val Asp Asp Ile Lys Ile Ser Ile Pro Asn Th #r Gln Pro Ile Tyr Asp            100       #           105       #           110Met Gly Asn His Arg Ser Glu Pro Ile Gln Pr #o Thr Gln Pro Gln Tyr        115           #       120           #       125Asp Met Pro Thr Ala Asn Asn Val Ala Ser Me #t Thr Leu Glu Gln Leu    130               #   135               #   140Glu Ala Gln Ser Gln Asn Val Gly Phe Asn Gl #y Ile Asn Ser Ser Ser145                 1 #50                 1 #55                 1 #60Pro Glu Leu Arg Val Gln Leu Ala Glu Leu Se #r His Glu Glu His Gln                165   #               170   #               175Val Asp Tyr Asn Leu Ser Phe Asn Glu Pro Ly #s Ala Glu Thr Thr Ala            180       #           185       #           190His Pro Lys Gln Thr Thr Gly Tyr Ile Gln Le #u Tyr Leu Ile Pro Lys        195           #       200           #       205Ser Ser Glu Glu Phe Asn Gly Ala Lys Leu Va #l Gln Ala Leu Glu Asn    210               #   215               #   220Leu Gly Phe Ile Leu Gly Lys Asp Glu Met Ty #r His Arg His Leu Asp225                 2 #30                 2 #35                 2 #40Leu Ser Val Ala Ser Pro Val Leu Phe Ser Va #l Ala Asn Leu Glu Gln                245   #               250   #               255Pro Gly Thr Phe Asn Ala Tyr Asn Leu Ala Gl #u Phe Asn Thr Ile Gly            260       #           265       #           270Ile Val Leu Phe Met Gln Leu Pro Ser Pro Gl #y Asn Asn Leu Ala Asn        275           #       280           #       285Leu Arg Met Met Met Arg Ala Ala His Thr Le #u Ala Glu Asp Leu Gln    290               #   295               #   300Gly Val Ile Leu Thr Glu Glu Gln Glu Ile Ph #e Asp Ala Asn Ala Glu305                 3 #10                 3 #15                 3 #20Gln Ala Tyr Leu Ala Arg Val                 325(2) INFORMATION FOR SEQ ID NO: 4:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 6 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: Not R #elevant          (D) TOPOLOGY: Not Relev #ant     (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #4: Ile Leu Ile Ile Val Gly1               5 (2) INFORMATION FOR SEQ ID NO: 5:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 10 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: Not R#elevant           (D) TOPOLOGY: Not Relev #ant    (ii) MOLECULE TYPE: peptide     (ix) FEATURE:          (A) NAME/KEY: Modified-sit #e           (B) LOCATION: 3          (D) OTHER INFORMATION:  #/note= “The peptide at this               location  #can be either Arg or Asn.”     (ix) FEATURE:          (A) NAME/KEY: Modified-sit #e           (B) LOCATION: 4          (D) OTHER INFORMATION:  #/note= “The peptide at this               location  #can be either Leu or Thr.”    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #5:Asp Leu Xaa Xaa Ile Leu Ile Ile Val Gly 1               5   #                10 (2) INFORMATION FOR SEQ ID NO: 6:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 13 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: Not R#elevant           (D) TOPOLOGY: Not Relev #ant    (ii) MOLECULE TYPE: peptide     (ix) FEATURE:          (A) NAME/KEY: Modified-sit #e           (B) LOCATION: 7          (D) OTHER INFORMATION:  #/note= “The peptide at this               location  #can be either”     (ix) FEATURE:          (A) NAME/KEY: Modified-sit #e           (B) LOCATION: 8          (D) OTHER INFORMATION:  #/note= “The peptide at this               location  #can be either Ile or Val.”     (ix) FEATURE:          (A) NAME/KEY: Modified-sit #e           (B) LOCATION: 10          (D) OTHER INFORMATION:  #/note= “The peptide at this               locaiton  #can be either Ile or Leu.”     (ix) FEATURE:          (A) NAME/KEY: Modified-sit #e           (B) LOCATION: 11          (D) OTHER INFORMATION:  #/note= “The peptide at this               location  #can be either Ile or Val.”    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #6:Ile Leu Ile Ile Val Gly Xaa Xaa Ala Xaa Xa #a Ala Leu1               5    #                10(2) INFORMATION FOR SEQ ID NO: 7:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 17 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: Not R #elevant          (D) TOPOLOGY: Not Relev #ant     (ii) MOLECULE TYPE: peptide    (ix) FEATURE:           (A) NAME/KEY: Modified-sit #e          (B) LOCATION: 7           (D) OTHER INFORMATION: #/note= “The peptide at this                location #can be either Ala or Leu.”     (ix) FEATURE:          (A) NAME/KEY: Modified-sit #e           (B) LOCATION: 8          (D) OTHER INFORMATION:  #/note= “The peptide at this               location  #can be either Ile or Val.”     (ix) FEATURE:          (A) NAME/KEY: Modified-sit #e           (B) LOCATION: 10          (D) OTHER INFORMATION:  #/note= “The peptide at this               location  #can be either Ile or Leu.”     (ix) FEATURE:          (A) NAME/KEY: Modified-sit #e           (B) LOCATION: 11          (D) OTHER INFORMATION:  #/note= “The peptide at this               location  #can be either Ile or Val.”     (ix) FEATURE:          (A) NAME/KEY: Modified-sit #e           (B) LOCATION: 14          (D) OTHER INFORMATION:  #/note= “The peptide at this               location  #can be either Ile or Leu.”    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #7:Ile Leu Ile Ile Val Gly Xaa Xaa Ala Xaa Xa #a Ala Leu Xaa Val His1               5    #                10   #                15 Gly(2) INFORMATION FOR SEQ ID NO: 8:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 6 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: Not R #elevant          (D) TOPOLOGY: Not Relev #ant     (ii) MOLECULE TYPE: peptide    (ix) FEATURE:           (A) NAME/KEY: Modified-sit #e          (B) LOCATION: 3           (D) OTHER INFORMATION: #/note= “The peptide at this                location #can be either Ile or Leu.”     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #8: Ala Leu Xaa Val His Gly 1               5(2) INFORMATION FOR SEQ ID NO: 9:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 8 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: Not R #elevant          (D) TOPOLOGY: Not Relev #ant     (ii) MOLECULE TYPE: peptide    (ix) FEATURE:           (A) NAME/KEY: Modified-sit #e          (B) LOCATION: 3           (D) OTHER INFORMATION: #/note= “The peptide at this                location #can be either Ile or Leu.”     (ix) FEATURE:          (A) NAME/KEY: Modified-sit #e           (B) LOCATION: 7          (D) OTHER INFORMATION:  #/note= “The peptide at this               location  #can be either Phe or Leu.”    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #9:Ala Leu Xaa Val His Gly Xaa Trp 1               5(2) INFORMATION FOR SEQ ID NO: 10:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 5 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: Not R #elevant          (D) TOPOLOGY: Not Relev #ant     (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #10: Tyr His Arg His Leu1               5 (2) INFORMATION FOR SEQ ID NO: 11:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 8 amino  #acids          (B) TYPE: amino acid           (C) STRANDEDNESS: Not R#elevant           (D) TOPOLOGY: Not Relev #ant    (ii) MOLECULE TYPE: peptide     (ix) FEATURE:          (A) NAME/KEY: Modified-sit #e           (B) LOCATION: 2          (D) OTHER INFORMATION:  #/note= “The peptide at this               location  #can be either Ala or Val.”     (ix) FEATURE:          (A) NAME/KEY: Modified-sit #e           (B) LOCATION: 6          (D) OTHER INFORMATION:  #/note= “The peptide at this               location  #can be either Leu or Val.”    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #11:Pro Xaa Leu Phe Ser Xaa Ala Asn 1               5(2) INFORMATION FOR SEQ ID NO: 12:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 4 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: Not R #elevant          (D) TOPOLOGY: Not Relev #ant     (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #12: Pro Gly Thr Phe 1(2) INFORMATION FOR SEQ ID NO: 13:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 4 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: Not R #elevant          (D) TOPOLOGY: Not Relev #ant     (ii) MOLECULE TYPE: peptide    (ix) FEATURE:           (A) NAME/KEY: Modified-sit #e          (B) LOCATION: 1           (D) OTHER INFORMATION: #/note= “The peptide at this                location #can be either Ile or Leu.”     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: #13: Xaa Phe Met Gln 1 (2) INFORMATION FOR SEQ ID NO: 14:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 7 amino  #acids          (B) TYPE: amino acid           (C) STRANDEDNESS: Not R#elevant           (D) TOPOLOGY: Not Relev #ant    (ii) MOLECULE TYPE: peptide     (ix) FEATURE:          (A) NAME/KEY: Modified-sit #e           (B) LOCATION: 1          (D) OTHER INFORMATION:  #/note= “The peptide at this               location  #can be either Ile or Leu.”     (ix) FEATURE:          (A) NAME/KEY: Modified-sit #e           (B) LOCATION: 5          (D) OTHER INFORMATION:  #/note= “The peptide at this               location  #can be either Val or Leu.”    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #14:Xaa Phe Met Gln Xaa Pro Ser 1               5(2) INFORMATION FOR SEQ ID NO: 15:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 33 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #15:ACAGAGATCC ATATGATGCA GGATTTGCGT CTG        #                  #         33 (2) INFORMATION FOR SEQ ID NO: 16:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 30 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #16:TTAACCAAGC TTAAGTGTAT CAGGCGTTGG          #                  #           30 (2) INFORMATION FOR SEQ ID NO: 17:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 28 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #17:AAGTCTCGAG GGCGTTGGCG TCTTTGAC          #                  #             28 (2) INFORMATION FOR SEQ ID NO: 18:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 8 amino  #acids          (B) TYPE: amino acid           (C) STRANDEDNESS: Not R#elevant           (D) TOPOLOGY: Not Relev #ant    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #18:Leu Glu Asp Pro Pro Ala Glu Phe 1               5(2) INFORMATION FOR SEQ ID NO: 19:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 53 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: Not R #elevant          (D) TOPOLOGY: Not Relev #ant     (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #19:Met Gly His His His His His His His His Hi #s His Ser Ser Gly His1               5    #                10   #                15Ile Glu Gly Arg His Met Asp Tyr Lys Asp As #p Asp Asp Lys Ala Arg            20       #            25       #            30Arg Ala Ser Val Glu Phe His Met Ala Ser Me #t Thr Gly Gly Gln Gln        35           #        40           #        45Met Gly Arg Gly Ser     50 (2) INFORMATION FOR SEQ ID NO: 20:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 14 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: Not R#elevant           (D) TOPOLOGY: Not Relev #ant    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #20:Met Ala Ser Met Thr Gly Gly Gln Gln Met Gl #y Arg Gly Ser1               5    #                10(2) INFORMATION FOR SEQ ID NO: 21:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 21 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: Not R #elevant          (D) TOPOLOGY: Not Relev #ant     (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #21:Met Gly His His His His His His His His Hi #s His Ser Ser Gly His1               5    #                10   #                15Ile Glu Gly Arg His             20 (2) INFORMATION FOR SEQ ID NO: 22:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 17 amino #acids           (B) TYPE: amino acid           (C) STRANDEDNESS: Not R#elevant           (D) TOPOLOGY: Not Relev #ant    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #22:Met Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ar #g Arg Ala Ser Val Glu1               5    #                10   #                15 Phe(2) INFORMATION FOR SEQ ID NO: 23:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 54 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: Not R #elevant          (D) TOPOLOGY: Not Relev #ant     (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #23:Met Gly His His His His His His His His Hi #s His Ser Ser Gly His1               5    #                10   #                15Ile Glu Gly Arg His Met Asp Tyr Lys Asp As #p Asp Asp Lys Ala Arg            20       #            25       #            30Arg Ala Ser Val Glu Phe His Met Ala Ser Me #t Thr Gly Gly Gln Gln        35           #        40           #        45Met Gly Arg Gly Ser His     50 (2) INFORMATION FOR SEQ ID NO: 24:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 31 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #24:GGAGGATCCC ATATGTTTGA ACCAATGGAA C         #                  #          31 (2) INFORMATION FOR SEQ ID NO: 25:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 31 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #25:TTCCGGTCGA CTCTTAATCA GCTTGCTTAC G         #                  #          31

We claim:
 1. A composition comprising a first purified protein, saidfirst purified protein comprising amino acid 39 through amino acid 328of SEQ ID NO. 2, the amino acid sequence of SEQ ID. NO. 2, the aminoacid sequence of SEQ ID NO. 3, or, in order, the amino acid sequences ofSEQ ID NO. 4, SEQ ID NO. 5, SEQ ID. NO. 6, SEQ ID. NO. 7, SEQ ID NO. 8,SEQ ID. NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO.13, and SEQ ID NO.
 14. 2. The composition of claim 1, further comprisinga second purified protein bound to said first protein.
 3. Thecomposition of claim 2, wherein said second protein is a cell divisionprotein of E. coli known as FtsZ, or a homolog thereof.
 4. Thecomposition of claim 3, wherein one of the proteins is fused to a T7.tagpeptide, a His-tag peptide, a Flag peptide, a heart muscle kinasesubstrate peptide, or combinations of said peptides.
 5. The compositionof claim 3, wherein one of said proteins is labelled with a reportermolecule.
 6. The composition of claim 5, wherein said reporter moleculeis fluorescent.
 7. The composition of claim 6, wherein said reportergrew molecule comprises fluorescein.
 8. The composition of claim 3,wherein one of said proteins is biotinylated.
 9. The composition ofclaim 4, wherein the sequence of the T7.tag peptide is set forth in SEQID. NO.
 20. 10. The composition of claim 4, wherein the sequence of theHis tag peptide is set forth in SEQ ID NO.
 21. 11. The composition ofclaim 4, wherein the sequence of the flag peptide and heart musclekinase substrate peptide is set forth in SEQ ID NO.
 22. 12. Thecomposition of claim 4, wherein one of the proteins is fused to apeptide whose sequence is set forth in SEQ ID NO.
 19. 13. Thecomposition of claim 4, wherein one of the proteins is fused to apeptide whose sequence is set forth in SEQ ID NO. 23.